Our long term goal is to delineate the molecular mechanisms by which osteoclasts contribute to bone metabolism with the expectation that this knowledge will guide more effective therapies to promote skeletal health. Ideally, new therapy targets to reduce osteoclast-mediated bone loss will block osteoclasts while also preserving bone formation. This may be challenging since evidence is mounting that osteoclasts, whether or not they are capable of resorbing bone, are needed for normal bone formation by osteoblasts. We have examined the vertebrae of mice in which the CD34 family member PODXL is deleted in hematopoietic/osteoclast lineage cells (hem lineage-PODXLdel mice). Bone density and osteoblast numbers were both elevated. Unexpectedly, the number and size of osteoclasts were also significantly higher. Thus, loss of PODXL in early osteoclast lineage cells reduces bone resorption while preserving osteoblast recruitment and bone formation. Our in vitro studies support that PODXL expression in osteoclast lineage cells reduces osteoclast differentiation. However, a relatively low level of PODXL expression is required for osteoclast actin ring formation and bone resorption. Our studies and the known functions of PODXL lead to our central hypothesis that early progenitor membrane expression levels of a PODXL membrane complex including CXCR4 and NHERF1 must be reduced to allow for precursor fusion and lower levels of this complex are required for activation of osteoclasts, enabling them to resorb bone. The following Specific Aims are designed to test this hypothesis 1. Determine the role of osteoclast lineage PODXL expression changes in skeletal acquisition and bone maintenance. We will comprehensively phenotype the hem lineage-PODXLdel mice, establish the effects of ovariectomy on bone metabolism in hem lineage-PODXLdel mice, define the impact of loss of PODXL in early osteoclast lineage cells during anabolic PTH treatment, and compare mice with PODXL deleted in late osteoclast lineage cells to the hem lineage-PODXLdel mice. 2. Interrogate the molecular mechanisms by which the PODXL, CXCR4, and NHERF-1 complex influences osteoclast lineage cells. We will identify structural domains of PODXL and NHERF1 involved in complex formation, membrane expression, differentiation, and internalization during differentiation, resolve the mechanism of down regulation of PODXL, CXCR4, and NHERF1 gene expression by M-CSF, and ascertain the influences of PODXL and binding partners on integrin interactions, actin ring formation, and osteoclast function. PUBLIC HEALTH RELEVANCE: Bone loss associated with aging and postmenopausal osteoporosis is due to excess osteoclast-mediated bone resorption. Excess bone loss leads to risks of debilitating fractures. An estimated 44 million Americans are at risk of osteoporosis. Within the U.S., 10 million people are estimated to already have this disease and an additional 34 million are suspected of having low bone mass and at risk of developing the disease. There are 1.5 million osteoporotic fractures in the U.S. each year and the cost of caring for these fractures is estimated at $12 billion to $18 billion. Indirect costs in lost productivity of patients and care givers add billions to this cost. Thus, therapies that target bone resorbing osteoclasts while preserving bone formation have the potential to have a significant impact on patients.